石刁柏雄性连锁的AFLP及SCAR标记的建立

目的:对石刁柏雌雄株基因组差异进行分析,筛选雄性或雌性连锁的分子标记.方法:利用限制性片段长度多态性技术,设计了多个引物组合,分别对石刁柏雌雄株基因组进行扩增.结果:在使用的72个引物组合中,引物组合E-AAG+M-CAT从雄性基因组中扩增出了一个雄性连锁的标记(MLDA555),该序列长度为555bp,AT含量为59%.Blast检索未发现相似序列.根据该片段序列设计的引物将该标记转化为雄性连锁的大小为523bp的稳定的SCAR标记,经过不同基因型雄性个体的验证证明该标记广泛存在于不同基因型石刁柏雄性个体中.结论:通过AFLP扩增筛选得到了石刁柏雄性连锁的AFLP和SCAR标记,为石刁柏性别决定机制的理解及石刁柏的分子标记辅助育种提供理论资料和技术支持.     

雌核发育鲢各系及与普通鲢生长的比较研究

在相同池塘养殖条件下,迸行了雌核发育鲢各系(当年鱼)及与普通鲢的生长对比试验。结果表明:所选育的雌核发育鲢1系、2系间无显著差异,杂交系较1系、2系呈一般显著性差异(P<0.05);雌核发育鲢较普通鲢生长快,平均体长增长快7％以上,体重增长快14％以上,呈显著差异(P<0.01);其中雌核发育鲢杂交生长最快,其体重增长比普通鲢快23.8％,体长增长比普通鲢快12.4％,呈非常显著差异(P<0.005)。密度越稀,增长越快,雌核发育鲢各系成活率(83.4％以上)均高于普通鲢(64.8％),且雌核鲢体型较普通鲢肥厚。     

Cloning and evolutionary analysis of cyclin B gene introns in cyprinids with different ploidy levels

Partial fragments of the cyclin B gene from triploid, tetraploid, and pentaploid hybrids of red crucian carp × blunt snout bream, blunt snout bream, grass carp, silver carp, and bighead carp were amplified. One DNA fragment was amplified from the blunt snout bream, grass carp, silver carp, and bighead carp (750, 950, 720, and 720 bp, respectively). Two fragments (1200 and 900 bp) were amplified from the red crucian carp, common carp, and allotetraploids. The triploid and pentaploid hybrids yielded three DNA fragments (1200, 900, and 750 bp). The 1200 bp fragment of the allotetraploid crucian carp, triploid, tetraploid, pentaploid hybrids of red crucian carp × blunt snout bream shared 99.5%, 98.9%, 99.5%, and 88.7% homology, respectively, with the maternal DNA. The 900 bp fragment shared 97.5%, 94.6%, 94.2%, and 89.9% homology, respectively. Our results suggest that inheritance is maternally dominated. Furthermore, we observed preferential elimination of the paternal sequences in the allotetraploid hybrids. Based on these sequence analyses we constructed a phylogenetic tree to explain the relationships among the different ploidy levels.     

Construction of AFLP-based genetic linkage maps for the Chinese shrimp Fenneropaeneus chinensis

Fenneropaeneus chinensis is an important species in marine fishery resources and aquaculture in China. A genetic linkage map is essential for improving the efficiency of its breeding by marker-as- sisted selection and identifying commercially important genes. Linkage maps of F. chinensis were constructed with an F2 mapping population (110 progenies) using amplified fragment length polymor- phic (AFLP) marker in this study. Fifty-five AFLP primer combinations produced 532 AFLP markers fitting for map strategy in mapping family. The markers with 3:1 segregating ratios were analyzed using F2 intercross model for the common linkage map, while the markers with 1:1 ratio were analyzed using the pseudo-testcross strategy. The maps of male, female and common were constructed. The female map included 103 markers that formed 28 linkage groups, covering a total length of 1090 cM. All mark- ers were linked with the linkage groups. Segregation distortion was observed for 6 of 103 markers in the female map. The average distance between markers was 14.53 cM and ranged from 4.4 to 24.8 cM. The male map included 144 markers that formed 35 linkage groups. Ten markers remained unlinked in male map. Segregation distortion was observed for 7 of 144 markers in the male map. The total dis- tance of male map covered 1617 cM. The average distance between markers was 16.36 cM. The male map was 32.6% longer than the female map, which may reflect sex-specific recombination rates in Chinese shrimp. The common map was composed of 216 markers, including in 44 linkage groups covering a total distance of 1772.1 cM. Two markers remained unlinked. No distorted markers of 216 markers were shown in the common map. The distance between markers was 10.42 cM. An average estimated genome size for the Chinese shrimp was 2420 cM, which was consistent with the relative size of the Penaeid genome. The distribution of AFLP markers was relatively even in chromosomes of Chi- nese shrimp maps. The linkage analysis presented in this work provided some insight into the level of polymorphism and genetic variation of Chinese shrimp.     

Molecular cloning of growth hormone receptor (GHR) from common carp(Cyprinus carpio L.)and identification of its two forms of mRNA transcripts

Growth hormone receptor (GHR) belongs tothehematopoietic receptor superfamily[1]. The action ofgrowth hormone (GH) in regulating growth[2],re-production[3]and i mmunity[4]has been elucidated.The binding of GHtothe GHRontarget tissues trig-gers a cascade of tyrosine and protein phosphorylationevents, which cul minates in the biological action ofGH[5 ,6]. Up to date GHR cDNAs have been clonedfrom many species[7—9],including various kinds ofmammalian ani mals ; avian of chicken and domest…     

Rapid establishment of pure lines of silver carp (Hypophthalmichthys molirix) and bighead carp (Aristichthys nobilis)

The diversity of gynogenetic, artificial sex reversal and natural silver carp and bighead carp is examined using randomly amplified polymorphic DNA (RAPD) method.All of the 187 bands are obtained and 19 (10.16%) of them are polymorphic in gynogenetic silver carp.Meanwhile 32 (15.61%) out of 205 bands are polymorphic in control group.In gynogenetic bighead carp a total of 232 bands are identified and 11 (4.74%) out of them are polymorphic, while 25 (10.37%) out of 241 bands are polymorphic in control group.The genetic distance of four populations is calculated and it is 0.102 and 0.023 for gynogenetic silver carp and gynogenetic bighead carp respectively.The values of natural silver carp and bighead carp are 0.161 and 0.104.From the UPGMA trees constructed based on genetic distance, the sex reversal individuals that match with the gynogenetic female individuals are picked out.A new breeding process of establishing a pure line is developed.     

Detection of Grass Carp Hemorrhage Virus （GCHV） from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as “Red Spot Disease“, is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and eomparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplifica-tion. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain in-duces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines,including CAB, FHM, CIK, EPC, CCO and G(X), infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHVstrains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and thetwo strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-Vviruses have been replicated and amplified despite there being no cytopathogenic eifects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published re-cently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp frag-ment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is i-dentical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously,the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product,and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.     

137 Cs-γ 辐照对实验红鲫 AFLP 多态性的影响

摘要:目的 观察137 Cs-γ 辐照对实验红鲫 AFLP 的变化,探讨不同剂量137 Cs-γ 辐照对实验红鲫 DNA 多态性的影响。 方法 根据前期实验结果,将实验红鲫 C1HD 系随机分为 5 组,10 尾 / 组,即空白对照组、1 / 16 LD50 、1 / 8 LD50 、1 / 4 LD50 、1 / 2 LD504 个辐照组,设置 1. 94 Gy、3. 88 Gy、7. 76 Gy、15. 53 Gy4 个辐照剂量组和一个空白对照组,用生物辐照仪对实验红鲫进行一次性辐照。 提取辐照前和辐照后的实验红鲫血液 DNA,经 AFLP-PCR 扩增,并进行多态性信息含量( PIC) 、基因杂合度( H) 、香农信息指数( I) 分析。 结果 实验红鲫在137 Cs-γ 辐照处理前的 PIC、H 和 I分别为 45. 20% 、0. 19 和 0. 28,经137 Cs-γ 辐 照 处 理 后 分 别 为 72. 27% 、0. 28 和 0. 41,且 遗 传 学 指 数 升 高。 结 论经137 Cs-γ 辐照处理后,实验红鲫的 AFLP 多态性信息含量增加,基因杂合度升高。 AFLP 标记实验红鲫 C1HD 系可用于监测核辐射污染。     

Construction of AFLP-based genetic linkage maps for the Chinese shrimp Fenneropaeneus chinensis

Fenneropaeneus chinensis is an important species in marine fishery resources and aquaculture in China. A genetic linkage map is essential for improving the efficiency of its breeding by marker-assisted selection and identifying commercially important genes. Linkage maps of F. chinensis were constructed with an F2 mapping population （110 progenies） using amplified fragment length polymorphic （AFLP） marker in this study. Fifty-five AFLP primer combinations produced 532 AFLP markers fitting for map strategy in mapping family. The markers with 3：1 segregating ratios were analyzed using F2 intercross model for the common linkage map, while the markers with 1：1 ratio were analyzed using the pseudo-testcross strategy. The maps of male, female and common were constructed. The female map included 103 markers that formed 28 linkage groups, covering a total length of 1090 cM. All markers were linked with the linkage groups. Segregation distortion was observed for 6 of 103 markers in the female map. The average distance between markers was 14.53 cM and ranged from 4.4 to 24.8 cM. The male map included 144 markers that formed 35 linkage groups. Ten markers remained unlinked in male map. Segregation distortion was observed for 7 of 144 markers in the male map. The total distance of male map covered 1617 cM. The average distance between markers was 16.36 cM. The male map was 32.6% longer than the female map, which may reflect sex-specific recombination rates in Chinese shrimp. The common map was composed of 216 markers, including in 44 linkage groups covering a total distance of 1772.1 cM. Two markers remained unlinked. No distorted markers of 216 markers were shown in the common map. The distance between markers was 10.42 cM. An average estimated genome size for the Chinese shrimp was 2420 cM, which was consistent with the relative size of the Penaeid genome. The distribution of AFLP markers was relatively even in chromosomes of Chinese shrimp maps. The linkage analysis presented in this work provided some insight     

Short-term dynamics of nutrients influenced by upwelling in a small oligotrophic coastal ecosystem, Gan Bay, in the northwest Philippines

We present a time-series analysis of nutrient and pCO2 (partial pressure of CO2) levels in an oligotrophic coastal ecosystem (Gan Bay), which was likely to be influenced by upwelled subsurface water. Gan Bay is off Currimao Harbor, in the northwest Philippines and is located at the boundary of the South China Sea (SCS). This 42-h time-series observation was conducted in December 2006. In addition to continuous observations of dissolved oxygen (DO) and pCO2, discrete samples were collected at a depth of 5 m every 3 h for measurements of nutrients, including soluble reactive phosphorus (SRP) and inorganic nitrogen (NO3 +NO2 ) in order to examine their dynamics and possible physical and biological controls. We observed remarkably large short-term variations in the surface water,spanning a 10-fold change for SRP (32–330 nM) and from <0.3 μM to 4.3 μM for NO3 +NO2 . DO also varied substantially from a lower end of 171 to 205 μM O2. Surface water pCO2 changed from an equilibrium stage with the atmosphere ( 386 μatm) to a stage where it was a significant source for the atmospheric CO2 (seawater pCO2 469 μatm). We found that the variation of nutrients was driven neither by tidal mixing nor by biological activities, as was suggested by the variations in the total bacterial abundance and chlorophyll a. Instead, our inverse T–S relationship suggested a two end-member mixing process during the observation period. The N:P ratio throughout the observation period was 13.2, which is characteristic of SCS subsurface and deep waters. Moreover, pCO2 was correlated inversely with the sea surface temperature. It is likely, therefore, that an upwelled subsurface cold water with high nutrients, low-temperature and high-pCO2 existed. It should be noted that this upwelled cold water did not appear to impact the entire observation period (approximately 35 h of 42 h), which might suggest an extremely dynamic nature for this upwelled cold water mass.     

Characteristics of lateral heterogeneities with thermal and chemical origins in the pyrolitic lower mantle

The relative changes between shear and compressional velocities (R_SP = ∂ ln V_S/∂ ln V_P), bulk sound and shear velocities (R_CS = ∂ ln V_C/∂ ln V_S), and density versus shear wave velocity (R_ρS = ∂ ln ρ/∂ ln V_S) in response to thermal and chemical variations were investigated for the pyrolitic lower mantle. For heterogeneities with thermal origins, R_SP increases from 1.7 to 2.0 together with R_ρS decreasing from 0.4 to 0.2 and R_CS = ∼0.27 from the top to the bottom of the lower mantle. In comparison, chemical variations (bulk iron or silica contents) are characterized by R_SP < 1.5 and R_CS > 0.5 at lower mantle depths. Negative values of R_ρS and R_CS are indicative of chemical anomalies in the lower mantle, but a combination of thermal and chemical heterogeneities may be required to produce velocity and density anomalies at the magnitudes observed in seismic data. Further refinement of these characteristics requires data on the higher order pressure and temperature derivatives of the elastic moduli of the constituent phases.     

上海地区汉族人单胺氧化酶基因微卫星DNA多态性研究

应用扩增片段长度多态性技术,分析单胺氧化酶基因的ＭＡＯＡ（ＣＡ）。力ＭＡＯＢＩ（ＧＴ）ｎ２个基因座的微卫星ＤＮＡ多态性。在２４６名（男性１２２人,女性１２４人）无血缘关系上海地区汉族人中检出ＭＡＯＡ（ＣＡ）ｎ基因座８－种等位基因（１１０－１２４ｂｐ）和１９种基因型。     

DNA分子标记技术在药用植物上的应用

药用植物是我国极其宝贵的自然资源之一,开展药用植物的研究具有重要的理论与现实意义.随着分子生物学的快速发展,DNA分子标记技术作为一种新的遗传标记技术越来越广泛地应用于药用植物研究的许多领域中.本文简要介绍了DNA分子标记在药用植物研究中一些常见技术(如RAPD、RFLP、AFLP、SCAR、ISSR、SNP等),同时对分子标记技术在药用植物种质资源鉴定、药用植物遗传多样性、药用植物亲缘关系鉴定等方面的应用进行了较详尽的阐述.最后,对DNA分子标记技术的应用前景进行了展望.     

Construction and characterization of a bacterial artificial chromosome library of rice 5460F

Thermo-sensitive genie male sterile (TGMS) rice has a number of desirable characteristics for hybrid rice production. Many studies have demonstrated that the sterility of TGMS rice is controlled by a single recessive gene. It has been mapped for the first time on chromosome 8 and namedtms 1. Several AFLP markers which tightly linked to thetms 1 gene have been identified recently. In order to develop a detailed physical map of thetms1 gene-encompassing region and finally clone thetms1 gene, a bacterial artificial chromosome (BAC) library of rice 5460F (the fertile mutant line of TGMS rice 5460S) using a modified vector pECBAC1 has been constructed. The constructed 5460F BAC library consists of 16 896 clones with an average insert size of 119 kb, which represents about 4.7 times rice haploid genome equivalents. Neither chloroplast nor mitochondrial DNA was detected from the library. The library was screened with three single copy sequence amplified fragment length polymorphism (AFLP) markers which tightly linked totms1 gene as probes and eight positive clones were identified.     

Sodium current of neurosecretory cells in the eyestalk from the shrimp Penaeus japonicus

The properties of the inward current of medulla terminalis-X-organ (MTXO) cells isolated from the Penaeus japonicus eyestalk were studied with the whole-cell clamp technique in the presence of Ca²⁺ and K⁺ channel blockers. The inward currents had a threshold at about −50 mV and peaked at −10 mV. The reversed potential (V_rev) was very close to V_Na, the theoretical Nernst equilibrium potential for Na⁺. V_rev followed V_Na when the external Na⁺ concentration was varied and the currents were entirely suppressed by 30 nM tetrodotoxin (TTX), indicating that it was carried by Na⁺. The smooth line of concentration-dependent inhibition of sodium currents by TTX represented the best fit with the Hill equation, yielding an IC₅₀ of 2.1 ± 0.1 nM. The values of the half-maximal activation voltage V_h were −20.6 ± 0.5 and −19.3 ± 0.5 mV, respectively, in the absence and presence of 2 nM TTX. TTX had no significant effect on the voltage dependence of steady-state activation and inactivation of I_Na. Taken together, the results suggest that the inward current recorded under our experimental conditions was carried by sodium ions flowing through fast voltage-dependent Na⁺ channels.     

RT—PCR检测草鱼呼肠孤病毒的方法研究

草鱼呼肠孤病毒（Grass carp reovirus,GCRV）为草鱼出血病的病原．本实验根据GenBank中GCRV和其他水生呼肠孤病毒毒株的第六基因片段,在其保守区设计了一对GCRV特异性引物,建立了快速检测GCRV的逆转录聚合酶链式反应（RT—PCR）方法．该PCR体系中,上下游引物的最适终浓度为120nmol／L,最适退火温度为52℃．PCR特异性试验表明：所设计的引物只能扩增GCRV的核酸,而不能扩增嗜水气单胞菌BSK-10、WSSV以及正常CIK细胞的DNA或RNA．敏感性试验表明,当GCRV的反转录模板稀释至5-7时,PCR结果还为阳性．用所建立的RT—PCR方法对5份样品进行检测,结果表明本研究建立的RT—PCR检测方法可靠且可行．     

罗汉果性别相关的RAPD标记

应用随机扩增多态性DNA(RAPD)技术寻找与罗汉果性别相关的分子标记,筛选了130个10 bp的随机引物,发现有4个引物(S60、S90、S100、S343)能在雌、雄株DNA间扩增出5条差异性片段,大小在300~1 300 bp之间,表明这些特异带可被用来作为性别鉴别的特异性分子遗传标记。